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Non-clinical safety, immunogenicity and anticancer efficacy of VXM06,
a live attenuated Salmonella Typhimurium oral T cell vaccine against WT1
Sébastien Wieckowski1, Lilli Podola2, Marco Springer2, Iris Kobl2, Zina Koob3, Caroline Mignard3, Alan Broadmeadow4,
Pauline Stevens4, Clare Chesher4, Amine A. Berkane5, Ming Wei5, Klaus M. Breiner1, Albrecht Meichle2,
Philipp Beckhove6, Marc Mansour1, Matthias Schroff1, Heinz Lubenau2
1VAXIMM AG, Basel, Switzerland; 2VAXIMM GmbH, Mannheim, Germany; 3Oncodesign S.A., Dijon, France; 4Envigo CRS Ltd, Huntingdon, United Kingdom; 5CellVax S.A.S., Romainville, France; 6Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany.
Background
VA M
VAXIMM's oral T-cell vaccine platform is based on the approved, live attenuated Salmonella Typhistrain Ty21a vaccine, which has been administered in millions of individuals for prophylacticvaccination against typhoid fever. This strain has been thoroughly studied, is safe and welltolerated. The bacteria are modified to deliver an eukaryotic expression plasmid, which encodesthe genetic information of a specific target antigen1.
Figure 2. Schematic representation of VXM06 oral T-cell vaccine.
Our lead vaccine VXM01 encodesvascular endothelium growthfactor receptor 2 (VEGFR2) inorder to evoke an immuneresponse specifically directedagainst the tumor vasculature. Itis currently in clinicaldevelopment as a treatment forvarious solid cancer types. Themurine analogue of VXM01 hasshown consistent anti-angiogenic activity in differenttumor models in several animalstudies2. A proposed mechanismof action of Salmonella Typhistrain Ty21a oral T-cell vaccinesis described in Figure 1.
Figure 1. Intra-lymphatic delivery of Salmonella Typhi strain Ty21a T-cell vaccines via the oral route leading to target-specific T-cell activation.
The current study summarizes the non-clinical safetyprofile, immunogenicity and preclinical anti-cancerefficacy for the Salmonella Typhimurium SL7207 murinevaccine VXM06 (Figure 2) which encodes a murineWilm's tumor 1 (WT1) protein variant lacking all thezinc fingers. The empty vector, i.e. without plasmid, wasused as negative control throughout the study.
Toxicology
The preclinical safety profile of VXM06 andthe empty vector control was assessed inC57BL/6J mice, with n=10♂ and 10♀ in
both the treatment period and recovery(R) phases, following oral gavageadministration of doses up to 108 colony-forming units (CFU) per occasion on days1, 3, 5, 7, and once monthly thereafterduring 13 weeks, followed by 6-weekrecovery, in a GLP-compliant toxicologystudy (Figure 3).
Figure 3. Design of the 13-week toxicology study consistingin a treatment period of 13 weeks followed by a 6-week recovery phase.
There was no evidence of proliferation of VXM06 in the feces or organs analyzed. High whiteblood cell values were measured in mice receiving empty vector or VXM06 at 106 or 108
CFU/occasion. These findings were considered to be a Salmonella vector effect. Importantly,there were no deaths related to empty vector or VXM06, and no clear treatment related clinicalsigns, nor bodyweight, food consumption, hematology, organ weight and macroscopic pathologyfindings. Of note, small reductions of plasma calcium and phosphorus concentrations in malesand females given VXM06, persisting to the end of the recovery period were reported.
Histopathology analyses revealed multifocal inflammation and single cell necrosis in the liver ofanimals treated with either the empty vector control or VXM06 at 108 CFU/occasion (Figure 4).This finding was attributed to the bacterial vector when given at the highest dose. There was noclear evidence of recovery for the liver changes following 6 weeks respite from treatment.
Figure 4. Histopathology findings observed in ≥2% of the animals examined, males and females pooled together, after 13 weeks of treatment with empty vector (grey and black stacked bars) and VXM06 (green stacked bars) at 108 CFU/occasion (left bars) and after 6-week recovery (R; right bars).
ImmunogenicityThe immunokinetic study was performed in healthy C57BL/6 mice (n=5 per group), vaccinated 4times every other day via the oral route with 1010 CFU of either VXM06 or the empty vectorcontrol. The frequency of antigen-specific T cells was measured at different time points in thespleen by flow cytometry using fluorescently labelled MHC class I/peptide pentamers, withoutprior in vitro stimulation (Figure 5A).
Figure 5. (A) Experiment design and treatment schedule, and (B) frequency of WT1 antigen-specific cells among CD8+ T cells in the splenocytes of healthy C57BL/6 mice treated with the empty vector (grey boxplots) and VXM06 (green) at the indicated time points after the final vaccination.
Vaccination with VXM06 induced a significant systemic CD8 T cell response specific to WT1antigens, with a peak immune response detected 7 to 10 days after the final vaccination(Figure 5B). Comparable immunogenicity kinetics were measured in healthy animals usingSalmonella Typhimurium vaccines encoding murine VEGFR2 and mesothelin proteins3,4.
Anticancer efficacyWe evaluated the prophylactic anticancer activity ofVXM06 in the FBL-3 disseminated model of leukemiaexpressing WT15. Empty vector and VXM06 were givenby oral gavage at a dose of 108 CFU on days 1, 3, 5and 7 as a prime vaccination, and on days 14 and 22as boosts (Figure 6). C57BL/6 mice (n=10) thenreceived 5×106 viable FBL-3 cells by intraperitonealinjection on day 20. All surviving animals were re-challenged with 5×106 viable FBL-3 cells byintraperitoneal injection on day 100.
1. Darji A. et al., Cell 1997; 91:765. 2. Niethammer AG. et al., Nature Medicine 2002; 8:1369. 3. Wieckowski S. et al., European Journal of Cancer 2016; 69:S106. 4. Wieckowski S. et al., Cancer Research 2017; 77(13S):Abstract nr 4558. 5. Kohrt HE. et al., Blood 2011; 118:5319.
References
Contact and Information
Sébastien Wieckowski, Ph.D.
VAXIMM AGSenior Scientist
Office: +41 61 633 29 56
Fax: +49 621 8359 687 99
www.vaximm.com
Technologiepark Basel
Hochbergerstrasse 60c4057 Basel
Switzerland
Poster No. B058 presented during the Cancer Vaccines and Targets session at the Third CRI-CIMT-EATI-AACR International Cancer lmmunotherapy Conference on September 8th 2017 in Mainz/Frankfurt, Germany.
Prophylactic vaccination with VXM06 was highly tolerated, as no deterioration in general statuswas observed during the treatment, and neither death nor significant body weight loss wererecorded during the prime/boost treatment (Figure 7A). Vaccination with VXM06 generated arapid and sustained anti-leukemia effect with 100% (10 out of 10) of surviving animals 80 daysafter leukemia challenge (P<0.0001), confirming the data from a previous experiment3,4. Incontrast, vaccination with the empty vector control did not show any anti-cancer activity, as themedian survival reached 41 days, and 0% (0 animals out of 6) of cancer regression wasobserved (Figure 7B). Importantly, 100% of surviving mice resisted re-challenge with FBL-3cells at least 100 days after leukemia re-challenge (P<0.0001), demonstrating that vaccinationwith VXM06m generated a potent memory T cell response against the leukemia (Figure 7B).
Figure 6. Experiment design and treatment schedule in the prophylactic and re-challenge experiment.
Figure 7. (A) Evolution of the bodyweight in each individual animal, and (B) overall survival in the differenttreatment groups. The blue arrows represent the time points of leukemia challenge. Treatment-naive animals(yellow curves) were used as a control for the FBL-3 rechallenge and received the leukemia cells only day 100.
We finally evaluated the therapeutic efficacy of VXM06in the FBL-3 model. C57BL/6 mice (n=8 per group)received 5×106 viable FBL-3 cells by intraperitonealinjection on day 0. Empty vector and VXM06 were thenadministered by oral gavage at a dose of 109 CFU ondays 1, 3, 5 and 7 as a prime vaccination, and on days14 and 21 as boosts (Figure 8). The overall survivalwas monitored up to day 94.Figure 8. Design of the therapeutic study.
Therapeutic vaccination with VXM06 was well tolerated, as neither clinical sign, neither deathnor significant bodyweight loss were recorded during the treatment period (Figure 9A).Therapeutic vaccination with VXM06 induced the full leukemia control, with 100% (8 out of 8) ofsurviving animals 94 days after leukemia challenge (P<0.0001). In contrast, treatment with theempty vector control did not show any anti-cancer effect (Figure 9B).
Figure 9. (A) Evolution of the bodyweight in each individual animal, and (B) overall survival in both treatmentgroups, in the therapeutic setting. The blue arrow represents the time point of FBL-3 challenge.
Conclusions
▪ VXM06 was well-tolerated, generated substantial immunogenicity in
healthy animals, and a potent memory T-cell response in animals
bearing FBL-3 leukemia.
▪ Vaccination with VXM06 induced a rapid and sustained anti-cancer
activity in the FBL-3 model of leukemia, in both the prophylactic and
therapeutic settings.
▪ This study provides further evidence that VAXIMM’s oral T-cell
vaccination platform can be used to stimulate anti-tumor immunity
against various tumor-associated antigens, including WT1.
▪ These data paved the way for advancing the development of VXM06
into clinical development, in particular in leukemia.
moderate
slight
minimal
normal
VX
M06
em
pty
vecto
r
Teste
s (a
troph
y, tu
bular)
Ute
rus
(hyp
erpl
asia,
glan
dular,
cystic)
R R
Liver
(inf
lam
mat
ion,
sing
le cell n
ecro
sis)
Kid
neys
(cas
ts, h
yalin
e)
Adre
nals (h
yper
plas
ia,
subc
apsu
lar,
foca
l)
Laryn
x (in
filtra
te
infla
mm
ator
y ce
ll)
Sal
ivar
y gla
nd, M
andibu
lar
(agg
rega
tes, ly
mph
oid)
Kid
neys
(infiltra
te
infla
mm
ator
y ce
lls, i
nter
stitial)
Gal
l bla
dder
(hya
linos
is)
Kid
neys
(bas
ophi
lia, t
ubular
)
Duoden
um (dila
tatio
n
Bru
nner
's g
land
s)
Har
deria
n gla
nds
(infiltra
te, i
nflam
mat
ory ce
ll)
Sto
mac
h (infiltra
te, i
nflam
mat
ory,
muc
osal, g
land
ular
region
)
Lungs an
d bro
nchi
(infiltra
te, i
nflam
mat
ory ce
ll)
Pan
crea
s
(infiltra
te, i
nflam
mat
ory ce
ll)
Sple
en (pigm
ente
d
mac
roph
ages
, inc
reas
ed)
0
10
20
30
40
To
tal n
um
ber
of
an
imals
exam
ined R R R R R R R R R R R R R R
D1,3,5,7 D14 D21
FBL-3
challenge
D20D205
FBL-3
rechallenge
D100
A B
0 40 80 120 160 200
0
20
40
60
Days after initiation of treatment
Perc
en
t b
od
yw
eig
ht
ch
an
ge o
ver
baselin
e
empty vector
VXM06
untreated (treatment-naive)
0 40 80 120 160 200
0
50
100
Days after initiation of treatment
Perc
en
t su
rviv
al
empty vector
VXM06
untreated(treatment-naive)
****
D1,3,5,7 D14 D21
FBL-3 challenge
D0D94
A B
0 20 40 60 80 100
0
50
100
Days after tumor challenge
Perc
en
t su
rviv
al
empty vector
VXM06
****
0 20 40 60 80 100
0
10
20
30
Days after tumor challenge
Perc
en
t b
od
yw
eig
ht
ch
an
ge o
ver
baselin
e
empty vector
VXM06
A B
% o
f W
T1
ep
ito
pe
-sp
ec
ific
ce
lls
am
on
g C
D8
+ T
ce
lls
D1, 3, 5, 7
D12/14/17
splenocytes
D28
5 d 7 d 10 d 14 d 21 d
empty vector
VXM06
D21
P=0.026
1
2
3
4
5
% W
T-1
ep
ito
pe
-sp
ecif
ic c
ells
am
on
g C
D8+
T c
ells
Days 1,3,5,7 134
TREATMENT PERIOD (13 weeks)
36 64 92
RECOVERY (6 weeks)
Haematology Blood chemistry Organ weight
Macropathology Histopathology Biodistribution
Clinical condition, bodyweight, food consumption, Irwin, shedding
Salmonella TyphimuriumSL7207 carrier
Eukaryotic expressionplasmid encodingmurine WT-1 variant
